(a) Field of the Invention
The present invention relates to a method for incorporating 3,4-dihydroxyphenyl-L-alanine (DOPA) into mussel adhesive protein, more particularly, to a method for producing DOPA-incorporated mussel adhesive protein comprising the steps of transforming tyrosine auxotroph so as to express mussel adhesive protein, and culturing the transformant in a DOPA-containing medium to express mussel adhesive protein.
(b) Description of the Related Art
Mussels, one of marine organisms, have been studied as a potential source of water-resistant bioadhesives as they produce and secrete specialized water-resistant adhesive protein, adhere tightly to solid surfaces such as rock underwater and are not influenced by wave impact or buoyancy of seawater. They adhere tightly to surfaces underwater using byssus secreted from the foot. At the end of each thread is an adhesive plaque containing water-resistant glue that enables the plaque to anchor to wet solid surfaces. Mussel adhesive proteins are known as a strong natural adhesive, and they exhibit about two times higher tensile strength than epoxy resin while having flexibility, compared to chemically synthesized adhesive. Mussel adhesive proteins can adhere to various surfaces such as plastics, glass, metal, Teflon and biomaterials, and the like, and they can be applied in medical fields such as adhesion of biotissues at surgery or adhesion of broken tooth, and the like, as they are non-toxic to human body and do not cause immune response. And, they are environmentally friendly as they are biodegradable.
Mussel adhesive proteins consist of 6 kinds of proteins, fp(foot protein)-1 to fp-6. Most of them contain high content of DOPA(3,4-dihydroxyphenyl-L-alanine) that is derived in the hydroxylation process of tyrosine residues, and in fp-3 and fp-5 adjacent to the adhesion side, DOPA residue content are confirmed to be highest. To the contrary, DOPA residue-deficient mussel adhesive protein analogues are known to have significantly reduced adhesion, and thus, it is assumed that DOPA performs a main function for adhesion to surfaces. And, DOPA residues are converted into DOPA o-quinone through an oxidation process, and the DOPA o-quinone causes cross-linking between adhesion proteins, thereby functioning for achieving strong adhesion and keeping adhesive proteins from being dissolved even underwater. Thus, DOPA residue content of mussel adhesive protein is closely related to adhesion property.
Currently, to obtain 1 g of naturally extracted adhesive material from mussels, about 10,000 mussels are required. Thus, despite mussel adhesive proteins have excellent properties, there are many limitations in the industrial use of naturally extracted mussel adhesive proteins. Particularly, fp-5 is expected to be applied as a strong surface adhesive as it exhibits highest DOPA content of 25 mol %, however, it is substantially impossible to obtain the amount for commercialization through extraction. Alternatively, studies on mass production of mussel adhesive proteins using gene recombination technology have been conducted, and it has been confirmed that mussel adhesive protein fp-5 containing 6×-Histidine can be mass-produced in E. coli, and can be separated and purified through Ni-NTA (Biofouling Vol. 27, No. 7, August 2011, 729-737 “Recombinant mussel adhesive protein fp-5 (MAP fp-5) as a bulk bioadhesive and surface coating material”).
However, since a post-translational modification mechanism does not exist in E. coli, recombinant mussel adhesive proteins mass-produced in E. coli have unmodified amino acid residues unlike naturally extracted mussel adhesive proteins. Thus, mussel adhesive proteins produced in E. coli are subjected to separate enzymatic and chemical treatments so as to modify tyrosine with DOPA. For example, tyrosinase is known to be a representative enzyme for in vitro modification of tyrosine to DOPA or DOPA o-quinone, and tyrosine residues of mussel adhesive proteins produced in E. coli are in vitro modified to DOPA using tyrosinase. However, since this method requires additional reaction, involves high enzyme cost, and exhibits low modification degree, the industrial application is limited in terms of efficiency and economical feasibility.
Accordingly, there is a demand for development of technology for incorporating DOPA into mussel adhesive protein.